In ordinary fluorescence microscopy, defocused images outside the depth of focus are superimposed on an image formed on the focal plane. This globally lowers the contrast of microscopic image, which makes determination of fluorescence intensity difficult.
Confocal microscopy offers several advantages over conventional microscopy. The shallow depth of field, generally about 0.5 to 1.5 .mu.m, of confocal microscopes allows information to be collected from a well defined optical section rather than from most of the specimen as in conventional light microscopy. Consequently, out-of-focus fluorescence is virtually eliminated, which results in an increase in contrast, clarity and detection.
In a point scanning confocal system, the microscope lens focus the laser light on one point in the specimen at a time, i.e., the focal point. The laser moves rapidly from point to point to produce a scanned image. Very little of the laser light falls on other points in the focal plane. Both fluorescent and reflected light from the sample pass back through the microscope. The microscope and the optics of the scanner compartment focus the fluorescent light emitted from the focal point to a secont point, called the confocal point. A pinhole aperature, located at the confocal point, allows light from the focal point to pass through to a detector. Light emitted from outside the focal point is rejected by the aperature. Accordingly, only the image near the focal plane inside the sample is obtained as a microscopic image.
In two-photon absorption excitation type laser scanning fluorescence microscopy, a laser beam forms an optical spot having a high energy density and the optical spot three-dimensionally scans the inside of a sample in the same manner as in confocal laser scanning fluorescence microscopy. Because of the arrangement, fluorescence due to excitation based on two-photon absorption appears only from a point where the optical spot is located inside the sample but no fluorescence due to excitation based on two-photon absorption appears from other portions. Therefore, there appears no defocused image other than one on the focal plane, which improves the contrast of the microscopic image.
Two-photon excitation is made possible by the combination of (a) the very high, local, instantaneous intensity provided by the tight focusing available in a laser scanning microscope, wherein the laser can be focused to diffraction-limited waist of less than 1 micron in diameter, and (b) the temporal concentration of a pulsed laser. A high intensity, long wavelength, monochromatic light source which is focusable to the diffraction limit such as a colliding-pulse, mode-locked dye laser, produces a stream of pulses, with each pulse having a duration of about 100 femtoseconds (100.times.10.sup.-15 seconds) at a repetition rate of about 80 MHz. These subpicosecond pulses are supplied to the microscope, for example by way of a dichroic mirror, and are directed through the microscope optics to a specimen, or target material, located at the object plane of the microscope. Because of the high instantaneous power provided by the very short duration intense pulses focused to the diffraction limit, there is an appreciable probability that a fluorophore (a fluorescent dye), contained in the target material, and normally excitable by a single high energy photon having a short wavelength, typically ultraviolet, will absorb two long wavelength photons from the laser source simultaneously. This absorption combines the energy of the two photons in the fluorophore molecule, thereby raising the fluorophore to its excited state. When the fluorophore returns to its normal state, it emits light, and this light then passes back through the microscope optics to a suitable detector.
The probability of absorption of two long wavelength photons from the laser source simultaneously is dependent upon the two-photon cross-section of the dye molecule. In U.S. Pat. No. 5,770,737, one of us describes asymmetrical fluorene-containing two-photon chromophores of the formula: EQU D--Ar--A
wherein the Ar core is ##STR5## wherein R.sub.1 and R.sub.2 are alkyl groups having 8 to 12 carbon atoms, and wherein R.sub.1 and R.sub.2 are the same or different, wherein D is an electron donor moiety selected from the group consisting of ##STR6## and wherein A is an electron acceptor moiety selected from the group consisting of ##STR7##
The most active dyes described in U.S. Pat. No. 5,770,737 incorporate an easily polarizable olefinic double bond in the backbone of the molecule. This olefinic bond, although it greatly increases the two-photon absorption (TPA) cross-section of chromophores, has limited thermal and photochemical stability, thus reducing the range of its utility. We have now discovered dyes with increased thermal and photochemical stability while maintaining the same level of two-photon activity.
Accordingly, it is an object of the present invention to provide new chromophores having large two-photon cross-sections and increased thermal and photochemical stability.
Other objects and advantages of the present invention will be apparent to those skilled in the art.